JPH0570435B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to monoclonal antibodies against human rough aura bodies. The monoclonal antibody of the present invention is useful for detecting Lafola's disease, glycogen storage disease type.

Conventional techniques Detection of Lafola disease and glycogen storage disease type is done by muscle biopsy,
It is performed pathologically by liver biopsy, etc.

Rough aura disease (myocunus epilepsy) and glycogen storage disease type are abnormal glycogen with long lateral branches ( This is a disease in which rough aura bodies (rough aura bodies) accumulate in the heart muscle, nerve cells, liver cells, skin, etc.

Most cases of Rough Aura disease occur in families, with onset occurring around puberty, seizures, myoclonic dementia, and
It is a syndrome that manifests clinical symptoms such as cerebellar symptoms and personality changes, and takes a progressive course, resulting in death due to general weakness after more than 10 years.

Glycogen storage disease type (Andersen's disease) is a syndrome in which growth defects occur shortly after birth, hepatomegaly, splenomegaly, and hypotonia appear, complications of ascites occur, and death occurs within two years.

Problems to be solved by the invention Diagnosis of Lafola disease, glycogen storage disease type, is performed by muscle biopsy,
Diagnosis is made pathologically by liver biopsy, etc., but α-amylase treatment, β-amylase treatment,
Requires complicated techniques such as PAS staining, and
Nothing is certain.

Establishing a monoclonal antibody that reacts with abnormal glycogen (Rough Aura bodies) but not with normal glycogen is expected to become an extremely effective means for diagnosing Rough Aura disease and glycogen storage disease type.

Means to Solve the Problems The present inventors immunized mice with myocardial homogenate of patients with Lafola disease or abnormal glycogen extracted from it to create hybridomas, which react with abnormal glycogen but do not react with normal glycogen. A monoclonal antibody was established and the present invention was completed.

So far, there have been no reports regarding monoclonal antibodies against such abnormal glycogen.

The present invention will be explained in detail below.

The present invention provides monoclonal antibodies that react with rough aura bodies. The monoclonal antibody of the present invention belongs to the IgG class, and specific examples include KM
There is one named -279.

The monoclonal antibody of the present invention can be obtained by immunizing a mammal with a myocardial homogenate of a patient with Lafola disease or with Lafola bodies extracted therefrom, and fusing the spleen cells of the immunized animal with the osteoma cells of the mammal to obtain a hybridoma. Select a hybridoma cell line that reacts with Rough Aura bodies from the cell lines, culture the cell line in a medium, or administer it into the peritoneal cavity of a mammal to turn it into ascites, and monoclone that reacts with Rough Aura bodies in the culture or ascites. The monoclonal antibodies can be obtained by accumulating monoclonal antibodies and collecting the monoclonal antibodies from the culture or ascites fluid.

The mammals to be immunized and the mammals to which the osteoma cells are immunized include mice, rats, cows, horses, and the like.

A specific method for producing the monoclonal antibody of the present invention using a mouse as the mammal is shown below.

(1) Preparation of spleen cells from immunized animals Mice are immunized with myocardial homogenate of patients with Rough Aura disease or abnormal glycogen extracted therefrom, and spleen cells are prepared from the mice.

Myocardial homogenate abnormal glycogen from a patient with Rough Aura's disease is prepared according to the method of Reference Example 1.

The immunization method is to administer 8- to 10-week-old BALB/c mice subcutaneously, intravenously, or intraperitoneally using an appropriate adjuvant [for example, Complete Freund's
Adjuvant) or aluminum hydroxide gel and Bordetella pertussis vaccine, etc.], administer 10 to 100 μg/animal of Rough Aura's disease patient myocardial homogenate or abnormal glycogen extracted from it.
Thereafter, the same amount of the antigen used for the initial immunization is administered 2 to 5 times every 1 to 2 weeks.

Four to seven days after each immunization, blood is collected from the fundus venous plexus and the antibody titer against abnormal glycogen in the serum is examined.

The antibody titer is measured by solid-phase enzyme immunoassay (enzyme immunoassay: Igaku Shoin, 1978) as follows.

A 96-well EIA plate (manufactured by Flow Laboratories) was filled with 10 to 50 μg/ml of abnormal or normal glycogen in phosphate buffer/serine (PBS: disodium phosphate 1.83 g, potassium phosphate 0.21 g, salt 7.65 g, Distilled water 1, PH7.2)
Dispense the solution into 50μ/well and leave it at 4°C overnight to coat the plate with the antigen. Next, 200 μl of 1% bovine serum albumin (BSA)-PBS is dispensed per well, and the binding residues on the bottom of the plate are blocked with BSA. After washing the above plate thoroughly with PBS,
As the first antibody, 50μ of serially diluted samples (mouse serum, hybridoma culture supernatant, purified antibody)
/ well and incubate at 4°C overnight or at room temperature for 3 to 4 hours.
Leave it for a while. After washing six times with PBS, a 400-fold dilution of a rabbit anti-mouse immunoglobulin-peroxidase conjugate (manufactured by DAKO) as a second antibody was dispensed at 100μ/well and left at room temperature for 2 hours. After washing with PBS, use the ABTS substrate solution [a solution of 550 mg of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium dissolved in 0.1 M citrate buffer (PH4.2). Using a solution to which 1μ/ml of hydrogen peroxide was added just before,
Color development is measured by absorbance at OD _415on .

Mice whose antibody titer against abnormal glycogen is more than 10 ³ times that of normal mouse serum (OD value at 415 nm) are used as a source of immunized animal cells.

In order to perform cell fusion, immunized mice were intraperitoneally administered 10 to 200 μg/mouse of myocardial homogenate or abnormal glycogen extracted from the myocardium of Lafola disease patients 3 to 4 days before the fusion treatment, and after booster immunization, the spleen and prepare splenocytes.

(2) Preparation of osteopathoma cells As osteopathoma cells, established cell lines obtained from mice are used. For example, 8-azaguanine-resistant mouse (BALB/c-derived) osteopathoma cell line P3-
X63Ag8-U1 (P3-U1) [Current Topics in Microbiology and Immunology
Immunology) 81 , 1-7 (1978)], P3-NSI/
1-Ag41 (NS-1) [European Journal of Immunology (European J.
Immunology), 6 , 511-519 (1976)], SP2/0
-Ag14 (SP-2) [Nature, 276 ,
269-270 (1978)], P3-X63-Ag8653 (653) [Journal of Immunology (J.
Immunology), 123 , 1548-1550 (1979)], P3−
X63−Ag8 (X63) [Nature, 256 ,
495-497 (1975)] etc. are used.

This cell line was grown in 8-azaguanine medium [RPMI-1640 medium with glutamine (1.5mM), 2-
In normal medium supplemented with mercaptoethanol (5 x 10 ^-5 M), dientamycin (10 μg/ml) and fetal calf serum (FCS) (10%), an additional 8-
medium containing azaguanine (15 μg/ml)]
Passage the cells into a normal medium 3 to 4 days before cell fusion to ensure a cell number of 2 x 10 ⁷ or more on the day of fusion.

(3) Cell fusion To mice immunized with (1), 10 to 200 μg/mouse of myocardial homogenate or abnormal glycogen from patients with Lafola's disease is administered intraperitoneally, and 3 to 4 days later, the spleen is removed and splenocytes are prepared. These splenocytes and the osteopathoma cell line obtained in (2) were thoroughly washed with MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) or PBS, and the cell number was determined to be 5 to 10:1. Mix and centrifuge. After discarding the supernatant and loosening the precipitated cell group, add a mixture of 1 to 4 g of polyethylene glycol (PEG1000 to 4000), 1 to 4 ml of MEM medium, and 0.5 to 1.0 ml of dimethylsulfoxide while stirring.
Add ml/ ¹⁰⁸ splenocytes and MEM medium after 0.5-10 minutes.
Add 0.5-3ml. MEM every 0.5-2 minutes thereafter
After adding 0.5 to 3 ml of medium several times, add 30 to 30 ml of MEM medium.
Add 60ml.

After centrifugation, discard the supernatant, loosen the cells gently, add 50 to 200 ml of normal medium, and gently suspend the cells using a volumetric pipette. Dispense this suspension into a culture plate by half volume/well, and add 3-7%
Incubate for 10-30 h at 35-40 °C in a _CO2 incubator. Add half volume/well of HAT medium to culture plate [hypoxanthine (10 ^-5 to 10 ^-3 M) in normal medium,
Add a medium containing thymidine (10 ^-6 to 10 ^-4 M) and aminopterin (10 ^-8 to ^{10 -7} M), and then
Incubate for 10-30 hours. After that, every 10 to 30 hours for 1 to 3 days, discard half the volume of the culture supernatant and add the same amount.
Add HAT medium and incubate with _CO2 for 35 min.
Incubate for 10-14 days at ~40 °C.

For holes where fused cells that have grown in colonies are observed, discard half the volume of the supernatant and add HT medium (HAT medium with aminopterin removed).
Add the same amount and then every 10 to 30 hours for 1 to 3 days.
Perform conversion to HT medium.

After culturing in HT medium for 3 to 4 days, a portion of the culture supernatant is taken and the anti-abnormal glycogen antibody titer is measured by the enzyme immunoassay method described above.

Cloning is repeated 2 to 4 times using the limiting dilution method for wells in which antibody titers have been found, and those in which stable antibody titers have been found are selected as anti-abnormal glycogen monoclonal antibody-producing hybridoma lines.

(4) Preparation of monoclonal antibodies Anti-abnormal glycogen monoclonal antibodies obtained in (3) were applied to 8-10 week old BALB/c mice treated with pristane (2,6,10,14-tetramethylpentadecane). Production hybridoma cells 2-4x
Inject 10 ^5-7 pieces/mouse intraperitoneally. Hybridomas turn into ascites cancer in 10 to 21 days. Ascitic fluid is collected from this mouse, centrifuged to remove solids, salted out with 50% ammonium sulfate and 40% ammonium sulfate, and dialyzed against PBS (PH7.2) for 1 to 2 days. This dialyzed fraction can be used for purification and quantification as a crudely purified monoclonal antibody.

If further purification is required, the column is passed through a DEAE-Sepharose column, Protein A-column, Sephacryl S-300 column, etc., and active fractions (IgG, IgM, or IgA fractions) are collected.

Determination of antibody isotype and subclass
It is performed by the Ouchterlony method (Introduction to Immunology Experiments, Biochemical Experiment Methods 15, published by Gakkai Publishing Center, p. 74, 1981).

Quantification of protein amount is calculated using the furin method and absorbance at 280 nm.

The hybridoma cell line KM-279, which produces the monoclonal antibody of the present invention, is
Collection of Animal Cell Culture (ECACC)
It has been deposited as ECACC No. 86070304 on July 3, 1986.

Example 1 (1) Preparation of immunized mouse splenocytes 8-week-old BALB/c female mice (Shizuoka Prefecture Laboratory Animal Agricultural Cooperative Association) were injected with 2 mg/mouse of aluminum hydroxide gel as an adjuvant and killed Bordetella pertussis vaccine (Chiba Prefecture Serum). Laboratory) 1 x 10 ⁹ cells/mouse and 100μ of Rough Aura disease patient myocardial homogenate or abnormal glycogen extracted from it as an antigen.
g/mouse was administered intraperitoneally for immunization.

Thereafter, 100 μg/mouse of cardiac homogenate of Roughora's disease patients or abnormal glycogen extracted therefrom was administered intraperitoneally every 1 to 2 weeks.
The second and subsequent immunizations were applied. After the third immunization,
Five to seven days after immunization, blood was collected from the fundus venous plexus, and the anti-aberrant glycogen antibody titer in the serum was examined by solid-phase enzyme immunoassay. Mice whose serum antibody titer against abnormal glycogen was ¹⁰³ times or more that of normal mouse serum were further given 100 μg/g of abnormal glycogen.
ml was administered intraperitoneally for booster immunization, and 3 days later, splenocytes were prepared from the mice and used for cell fusion.

(2) Preparation of mouse osteopathoma cells 8-Azaguanine-resistant mouse osteopathoma cells P3-
U1 in normal medium [RPMI-1640 with glutamine
1.5mM, 2-mercaptoethanol 5 x 10 ^-5 M,
Dientamicin 10μg/ml and fetal bovine serum 0.1
ml/ml of culture medium] (37°C, 5% CO ₂ aeration), and after 4 days, 2×10 ⁷ or more cells were obtained.

(3) Preparation of hybridomas 1 x 10 ⁸ immunized mouse splenocytes and 2 x 10 ⁷ mouse osteoma cells thoroughly washed with MEM (manufactured by Nissui Pharmaceutical Co., Ltd.)
Mix and centrifuge at 1200 rpm for 5 minutes.

After thoroughly loosening the mixed cell group of splenocytes and P3-U1 obtained as a precipitate, the mixture was incubated at 37°C with stirring.
Polyethylene glycol-1000 (PEG-1000) 2
g, 0.5 mixture of 2 ml of MEM medium and 0.7 ml of DMSO
1 ml of MEM was added after 1 minute. after that
Add 1 ml of MEM 5 times every minute, then add MEM to make a total volume of 50 ml. After centrifugation at 900 rpm, discard the supernatant and gently loosen the cells.
HAT medium [hypoxanthine added to the normal medium above]
Add 100 ml of medium containing 10 ^-4 M, thymidine 1.5 x 10 ^-5 M, and aminopterin 4 x 10 ^-7 M,
Gently suspend the cells with a 10ml volumetric pipette.

The suspension was dispensed at 200 μg/well into a 96-well culture plate (falcon, manufactured by Becton-Ditzkinson), and incubated at 37°C in a 5% CO ₂ incubator for 10 to 30 minutes.
Incubate for 14 days.

Discard 100μ of the supernatant from the wells containing the fused cells that have grown in the form of colonies, and transfer to the HT medium [described above].
HAT medium with aminopterin removed]
Add 100μ and incubate at 37℃. After that, the medium was changed to HT medium in the same way for 2 days, and the culture was continued after 4 days.
A portion of the culture supernatant is collected, and the anti-abnormal glycogen antibody titer is measured by the solid-phase enzyme immunoassay method described above.

For the holes in which antibody titers were found, cloning was repeated twice using the limiting dilution method, and the clones in which stable antibody titers were found were used as anti-abnormal glycogen monoclonal antibody-producing hybridoma lines.
Select KM-279.

(4) Purification of monoclonal antibodies Pristane treatment [0.5 ml/mouse of 2,6,10,14-tetramethylpentadecane was administered intraperitoneally,
Breed for 2 weeks. 4×10 ⁶ cells/mouse of each of the hybridoma lines obtained above are intraperitoneally injected into 8-week-old female nude mice (BALB/c nu ⁻ /nu ⁻ ). After 10 to 21 days, the hybridoma strain turns into ascites cancer. After 10 to 21 days, ascitic fluid (4 to 10 ml/mouse) was collected from mice with accumulated ascitic fluid, and the solid content was removed by centrifugation. Ammonium sulfate salting out supernatant 50%, 40%
Salt out with ammonium sulfate and dialyze against PBS (PH7.2) for 2 days. This is used as a crudely purified monoclonal antibody. The crudely purified monoclonal antibody is passed through a DEAE-Sepharose column, eluted, and the IgG fraction is collected to obtain the purified monoclonal antibody.

Pathological diagnosis of Lafola's disease and glycogen storage disease type can be performed by conventional immunohistological techniques. A specific example is shown in Example 2.

(5) Antigen specificity of monoclonal antibodies The specificity of purified monoclonal antibodies was examined by solid-phase enzyme immunoassay. Antigens include abnormal glycogen derived from Rough aura disease myocardium (Reference Example 1);
Normal glycogen (manufactured by Hanui Chemical Co., Ltd.) and bovine serum albumin (manufactured by Sigma) were used.

The results are shown in Table 1.

Table 1 Reaction specificity of KM-279 Antigen Reactivity OD _{415onAbnormal} glycogen 0.990 Antibody glycogen 0.007Bovine serum albumin 0.008Example 2 Formalin of myocardium and skin from patients with Roughora's disease or glycogen storage disease type sliced into 5 μm pieces using a microtome Fixed paraffin-embedded tissue sections were fixed on ovalbumin-coated glass slides,
After deparaffinizing with xylene, it was made hydrophilic in stages with alcohol-water. Rinse with resin water for 5 minutes,
Endogenous peroxidase was blocked by standing for 30 minutes at room temperature in methanol containing 0.3% H ₂ O ₂ . Next, the sections were washed with PBS for 20 minutes and then left in diluted normal horse serum for 20 minutes at room temperature. Excess serum was aspirated from the sections, and the first antibody (anti-abnormal glycogen monoclonal antibody KM-297, 20 μg/ml) was added for 30 min.
Allowed to react for minutes. After washing, diluted biotinylated antibody (biotinylated rabbit anti-IgG antibody) was reacted for 30 minutes, and further after washing, avidin-biotin-peroxidase complex (manufactured by Vector) was reacted for 30 minutes. After thorough washing, the peroxidase substrate [0.1% diaminobenziidine tetrahydrochloride (diaminobenziidine) prepared in 0.1 M Tris-HCl buffer ( _PH7.2 ) containing 0.02% _H2O2 was added.
tetrahydrochloride] for 2 minutes, and the reaction was stopped under ice cooling. After staining with hematoxylene, the specimen was dehydrated with alcohol-water and xylene, fixed with Canada balsam, and examined under a microscope.

As a result, abnormal glycogen deposits (Rough Aura bodies) were observed extensively in the myocardium and skin of patients with Rough Aura disease and glycogen storage disease type.

On the other hand, no staining was observed in myocardial and skin sections from healthy subjects even after the same treatment.

Reference Example 1 5 to 10 g of myocardium derived from a patient with Rough Aura's disease is cut into small pieces, ground while adding 20% trichloroacetic acid solution to a final concentration of 10%, and then homogenized using a homogenizer. Homogenate at 3000rpm
Centrifuge for 10 minutes at
Add 99.5% ethanol and 1/30 volume of saturated potassium chloride solution and stir well.

Centrifuge again at 3000 rpm for 10 minutes and collect the precipitate fraction. This precipitated fraction is dissolved in resin water. The precipitate operation using ethanol and saturated potassium chloride was repeated three times and used as abnormal glycogen.

Effects of the Invention According to the present invention, a monoclonal antibody useful for detecting Lafola's disease or glycogen storage disease type is provided.

Claims (1)

[Scope of Claims] 1. A monoclonal antibody that reacts with Lahuaura corpuscles. 2. The monoclonal antibody according to claim 1, which belongs to the IgC class. 3. Immunize a mammal with a myocardial homogenate from a patient with Rough Aura disease or Rough Aura bodies extracted therefrom, fuse the spleen cells of the immunized animal with osteoparomatous cells of the mammal, and obtain Rough Aura bodies from the resulting hybridoma cell line. A reactive hybridoma cell line is selected, and the cell line is cultured in a culture medium or administered into the peritoneal cavity of a mammal to form ascites, and monoclonal antibodies that react with Rough aura bodies are accumulated in the culture or ascites. The monoclonal antibody according to claim 1, which is obtained by collecting the monoclonal antibody from culture or ascites.